Hops, the cones of the female hop plant (Humulus lupulus L.), are a key ingredient in beer and responsible for the bitter taste of this widely consumed beverage. Compounds from hops also have biological activities that may make them useful as pharmaceuticals or nutraceuticals, or leads for the development of pharmaceutical drugs (Zanoli and Zavatti, 2008). Although hop contains a wide range of phytochemicals, including polyphenols, stilbenes, essential oils (monoterpenes and sesquiterpenes) (Verzele, 1986), it is the terpenophenolics that are the most important for beer brewing and also have potential as medicinal agents (Verzele, 1986). Terpenophenolics may also be called prenylated polyketides. The terpenophenolics in hop can be divided into the prenylated acylphloroglucinols (most commonly called bitter acids) and the prenylflavonoids. The prenylated acylphloroglucinols include the alpha-acids (humulone, cohumulone and related compounds) and the beta-acids (lupulone, colupulone and related compounds) (FIG. 1). The alpha-acids isomerize during the brewing process giving rise to the bitter isohumulones. Several biological activities have been attributed to the humulones (Shimamura et al., 2001; Yajima et al., 2004; Lee et al., 2007) and lupulones (Lamy et al., 2007; Siragusa et al., 2008).
The main prenylflavonoid in hop is xanthohumol but related compounds such as desmethylxanthohumol, xanthogalenol and isoxanthohumol are also present (FIG. 2). Xanthohumol possesses a range of biological activities, which include antioxidation, cytoprotection via phase 2 protein induction and anticancer activities (reviewed in Stevens and Page, 2004; Goto et al., 2005; Colgate et al., 2006). The immediate metabolic precursor of xanthohumol, desmethylxanthohumol, isomerizes during brewing to form 6-prenylnaringenin and 8-prenylnaringenin. 8-Prenylnaringenin is the most potent phytoestrogen thus far identified (Milligan et al., 2000).
The biosynthetic pathways leading to the terpenophenolics in hop follow a common catalytic pattern consisting of three phases: polyketide formation through the action of a polyketide synthase, aromatic prenylation and cyclization/decoration (Page and Nagel, 2006). The proposed biosynthetic pathways leading to the major bitter acids and xanthohumol are shown in FIGS. 3 and 4, respectively.
The type III polyketide synthase responsible for the formation of the acylphloroglucinol core of the bitter acids compounds has been identified. Paniego et al. purified and cloned valerophenone synthase (VPS) (also called phlorisovalerophenone synthase) from hop (Paniego et al., 1999) (FIG. 3). The enzyme uses isovaleryl CoA or isobutyryl CoA as primers for polyketide formation. VPS gave phlorisovalerophenone (PIVP), which is the precursor for humulone and lupulone, when supplied with isovaleryl CoA and malonyl CoA. Similarly, VPS catalyzed the condensation of isobutyryl CoA and malonyl CoA to yield phlorisobutyrophenone (PIBP), the precursor for cohumulone and colupulone. The second phase of bitter acid biosynthesis involves prenylation of PIVP and PIBP. Prenylation of PIVP with one dimethylallyl diphosphate (DMAPP) molecule yields prenyl-PIVP and a second prenylation gives diprenyl-PIVP (also called deoxyhumulone). Prenylation of PIVP with three DMAPP molecules yields lupulone. The aromatic prenyltransferase(s) that carry out these reactions have not been identified. Zuurbier and co-workers showed that protein extracts from hop cones were capable of forming prenyl-PIVP, prenyl-PIBP, deoxyhumulone and deoxycohumulone from DMAPP and PIVP or PIBP (Zuurbier et al., 1998). The oxidase that converts deoxyhumulone to humulone has also not been identified at the gene or protein level.
The first step in prenylflavonoid biosynthesis is the condensation of p-coumaroyl CoA with three molecules of malonyl CoA to give chalconaringenin (also called naringenin chalcone), a reaction catalyzed by the type III polyketide synthase enzyme chalcone synthase (FIG. 4). Aromatic prenylation of the A ring of chalconaringenin with DMAPP yields desmethylxanthohumol, which is subsequently methylated at the 6′-hydroxyl group to form xanthohumol. Our group has recently identified the O-methyltransferase enzyme that performs this reaction (Nagel et al., 2008).
As discussed, only three genes encoding enzymes in hop terpenophenolic biosynthesis are known: i) valerophenone synthase, which catalyzes the formation of the polyketide moiety of bitter acid biosynthesis (Paniego et al., 1999), ii) chalcone synthase which forms the polyketide moiety of xanthohumol (Matousek et al, 2002) and iii) desmethylxanthohumol O-methyltransferase, which methylates desmethylxanthohumol to yield xanthohumol (Nagel et al., 2008). The aromatic prenyltransferase(s) participating in both of these pathways are not known.
As noted above, the genes encoding aromatic prenyltransferase enzyme(s) participating in either the bitter acid or prenylflavonoids pathways are unknown. However, several aromatic prenyltransferases involved in other branches of plant metabolism have been identified. These include a prenyltransferase that geranylates hydroxybenzoic acid in the shikonin biosynthetic pathway (Yazaki et al., 2002), a homogentisic acid prenyltransferase from Arabidopsis (Collakova and DellaPenna, 2001) and a recently discovered flavonoid prenyltransferase from Sophora flavescens (Sasaki et al., 2008).
Hop terpenophenolics are valuable plant-derived natural products. Enhanced production of bitter acids, xanthohumol or other hop terpenophenolics such as desmethylxanthohumol could be achieved though breeding and selection programs as well as genetic engineering with the use of genes encoding enzymes of the terpenophenolic biosynthetic pathways. In addition, the biosynthetic pathways leading to these metabolites may be transferred to bacteria, yeast, fungi or other prokaryotic or eukaryotic organisms to engineer terpenophenolic production in these hosts. Enhancing terpenophenolic levels in hop plants, engineering their synthesis in other plants or transferring their biosynthesis to microorganisms such as yeast are possible routes to producing greater quantities of these metabolites for use by the brewing industry, as pharmaceuticals or for other purposes. In order for the metabolic engineering of hop terpenophenolics to be achieved, genes encoding the enzymes of terpenophenolic biosynthesis must be identified.
There remains a need in the art to identify aromatic prenyltransferase enzymes, and nucleotide sequences encoding such enzymes, that catalyze the transfer of prenyl groups.